Page 295 - IJB-10-6

International Journal of Bioprinting 3D-Printed Zn/MgHA-PCL for angio/osteogenesis

2.3.5. In vitro osteogenic differentiation 2.4. In vivo experiments
potential evaluation
Composite scaffolds and BMSCs were co-cultured in a 2.4.1. Surgical procedure of animal studies in vivo
24-well plate at 1 × 10 cells/well. After cell attachment to In vivo bone repair was detected using a rat femoral defect
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the bottom of the plate, the culture media was replaced model. Forty-eight Sprague-Dawley rats (250 g; six weeks
with the composite scaffold extracts. After 7 and 14 days old; male) were divided into six groups: control, HA-PCLs,
of culture, cells were stained using the ALP kit (YEASEN, 10Zn@HA-PCLs, 5Mg10Zn@HA-PCLs, 10Mg10Zn@
China), and the optical density (OD) value at 405 nm was HA-PCLs, and 15Mg10Zn@HA-PCLs; the control group
determined using the enzyme marker method on day 7 of had femoral defect without a scaffold. After anesthesia
culture for semi-quantitative analysis. was induced with an intraperitoneal injection of 4%
pentobarbital sodium (0.9 mL/100 g), a hole with a depth
Mineralized nodules on BMSCs after co-culture were of 3 mm and a diameter of 3 mm was drilled into the lateral
observed using Alizarin Red S (ARS) staining. Briefly, after epicondyle using a hand drill. Utilizing 4-0 silk sutures, the
21 days of co-culture, cells were stained using ARS staining skin incision was stitched shut. The experiment followed
solution (C0138; Beyotime, China) and imaged using the recommendations of the Institutional Animal Care and
an optical microscope (Olympus, Japan). Subsequently, Use Committee (IACUC) of Shanghai Jiao Tong University
10% cetylpyridinium chloride solution was added for (animal protocol number: O_A2023001).
decolorization for 15 min. The OD value at 562 nm was
determined using an enzyme marker at 562 nm for 2.4.2. Histological evaluation
semi-quantitative analysis. and immunohistochemistry
The animals were divided into six groups, every group had
The RT-qPCR was performed to evaluate the expression eight animals. At week 6, half of every group was euthanized.
of osteogenic-related genes, including OPN, OCN, ALP, At week 12, the remaining animals were euthanized.
RUNX2, and β-actin. Briefly, after co-culturing sterile
composite scaffolds with BMSCs for 7 days, cDNA synthesis The femora were removed and fixed in 4%
and amplification were performed as described in Section paraformaldehyde for 7 days (replaced twice with tissue
2.3.4. The sequences of the primers are summarized in fixative). The tissue samples were subsequently decalcified
Table S1, Supporting Information. The experiments were with 10% EDTA for approximately 30 days, in preparation
repeated four times. for the histopathological analysis. According to the steps
specified by the manufacturer, hematoxylin and eosin
2.3.6. Western blot (H&E), Masson’s trichrome staining, and α-SMA, CD31,
Both HUVECs and BMSCs were cultured for 24 h and 14 and COL I immunohistochemical staining were performed.
days in composite scaffold extracts, respectively. The cells The sections were examined on a pathological tissue section
were then lysed on ice for 30 min in 200 μL of RIPA lysis scanner (Pannoramic MIDI II; 3DHIDTECH®, Hungary).
buffer. After that, the samples were centrifuged at 14,000 ×
g at 4°C for 15 min. The supernatants were collected and the 2.5. Statistical analysis
protein concentrations were measured using a BCA protein All data are presented as the mean ± standard deviation.
assay kit (Beyotime, China). The samples were separated Differences between the groups were examined by using
using a Precast Protein Plus Gel (36250ES10; YEASEN, Tukey’s post hoc test and one-way analysis of variance
China) and were then transferred onto a polyvinylidene (ANOVA). Statistical analysis was performed using
difluoride (PVDF) membrane. The membranes were GraphPad Prism software. A p-value < 0.05 (*p < 0.05, **p
blocked in TBST solution with 5% bovine serum albumin < 0.01, ***p < 0.001) was considered statistically significant.
(BSA; Beyotime, China). The membranes for HUVECs were
incubated overnight with anti-CD31 (1:2000; Proteintech, 3. Results and discussion
China), anti-VEGF (1:1000; Proteintech, China), and anti- 3.1. Preparation and characterization of element-
GAPDH (1:1000; Proteintech, China) antibodies. The doped HA
membranes for BMSCs were incubated overnight with In this study, HA and element-doped HA were synthesized
anti-COL1 (1:500; Proteintech, China), anti-OPN (1:1000; using elements Zn and Mg, as well as the H6L small
Proteintech, China), anti-OCN (1:500; ABclonal, China), molecular template agent, via the hydrothermal synthesis
and anti-GAPDH (1:1000; Proteintech, China) antibodies. method. The SEM results (Figure 2A) indicated that all HA
Thereafter, the membranes were incubated with an HRP- groups exhibit spherical shapes with diameters ranging
conjugated secondary antibody. The band signals were from 5 to 10 μm. Compared with HA synthesized without
detected using a chemiluminescence imaging system H L, the addition of H L resulted in the uniformly
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(Tanon, China). spherical HA with regular morphology. The sphericality
Volume 10 Issue 6 (2024) 287 doi: 10.36922/ijb.4243